Exploring the compatibility theory of traditional Chinese medicine formulae: the disassembled prescriptions study

Xue Yang,Xin-Yuan Luan

1Department of Medical Oncology, Tianjin Medical University General Hospital, Tianjin 300052, China. 2Department of Hepatobiliary and Pancreatic Surgery,Tianjin Hospital of ITCWM NanKai Hospital,Tianjin 300100,China.

Abstract Traditional Chinese medicine (TCM) has become very popular in the treatment of complex diseases worldwide in recent decades. Despite the promising performance of TCM, how herbs work synergistically in a formula and their therapeutic targets remain ambiguous.This largely limits the correct use and modernization of TCM formulas clinically. The study of disassembled prescriptions is important for investigating the compatibility theory of Chinese medicines. By dissecting the mechanisms of TCM formulae, the compatibility principles of some well‑known formulae, such as Realgar‑Indigo naturalis, have been elucidated to a certain extent. In this review, several common methods of disassembled prescription studies are summarized, including those of single herb or mineral,sub‑prescription, active ingredients or effective parts, large formulae, and systems pharmacology studies,as well as the methods of grouping based on the properties or efficacy of Chinese medicines, in order to provide evidence for formulae study. Moreover, some challenges to be addressed in disassembled prescriptions studies have been identified in this review.

Keywords: compatibility theory of formulae; traditional Chinese medicine; disassembled prescriptions study; systems pharmacology

Background

According to the theory of traditional Chinese medicine (TCM), the human body is a holistic being in which each organ or specific physiological part is interrelated. Under the guidance of the holistic concept, ancient TCM practitioners believed that different organs interacted when people suffered from an illness. Therefore, a combination of plant species/minerals targeting multiple etiological factors simultaneously, called formulae, increases in response to proper time and conditions. Modern medicine also uses combination therapy for the treatment of diseases, especially complex diseases,such as cancer, diabetes, and cardiovascular disease [1–3].Combination therapies that use multiple drugs are thought to be more effective than monotherapies because of their synergistic action or ability to overcome resistance under certain circumstances. However,TCM formulae differ from those of combination therapies used in modern medicine. First, herbs used in formulae are complex, with numerous unknown ingredients instead of a single chemical composition. Second, TCM formulae always include some herbs for the prevention of disease development according to disease transmission rules,such as“see the liver disease, know it will result in the dysfunction of the spleen, so add herbs to strengthen the function of the spleen”. Third, it is well known that the formation of any TCM formula consisting of various herbs is not random. Instead, TCM formulae are grouped based on certain combinational rules.

TCM formulae have been used to treat a variety of diseases dominantly or complementally for more than 2,500 years[4].Ancient Chinese medicine experts put forward a series of compatibility theories of Chinese medicines, such as “compatibility of seven relations” (compatibility of two herbs or minerals),“Jun‑Chen‑Zuo‑Shi” (In traditional Chinese medicine, the concepts of“Jun”, “Chen”, and “Zuo Shi” are often used to classify herbs and other natural substances according to their relative strength and potential effects on the body. “Jun” herbs are considered to be the most potent and are typically used to treat serious or chronic conditions. These herbs may have strong therapeutic effects, but can also potentially have stronger side effects. “Chen” herbs are considered to be less potent than “Jun” herbs and are often used to treat milder or acute conditions. These herbs tend to have milder therapeutic effects and fewer side effects. “Zuo Shi” herbs are considered to be the weakest and are typically used to support the action of other herbs or to treat minor or temporary conditions. These herbs tend to have minimal therapeutic effects and few or no side effects. In traditional Chinese medicine, practitioners may use a combination of herbs from these different categories to address different aspects of a condition and achieve a balanced treatment effect.), “the meridian tropism of herbs or minerals” and “the 18 incompatible medicaments, the 19 medicaments of mutual restraint”(contraindication of drug compatibility). Guided by these TCM theories, the construction of a prescription is always designed to contain different types of herbs or minerals to improve therapeutic efficacy and reduce side effects [5]. Generally, multiple active ingredients target multiple targets and exert a systemic effect [6].However, the compatibility principles, complex constitutions, and unknown action mechanisms of TCM prescriptions have hindered the modernization and acceptance of TCM by Western medicine.

The study of disassembling prescriptions is an important part of modern research on TCM prescriptions. In general, it refers to gradually subtracting one or several herbs or minerals from a prescription in order to observe the change in the curative effect or even look for effective components.Disassembling prescription studies are helpful for clarifying the scientific rationality of prescription compatibility, identifying major medicines or active ingredients,searching for the best compatibility ratio, and simplifying large complex formulae. In this review, seven primary research ideas and methods of disassembled prescriptions studies are summarized to provide evidence for the importance of studying the formulae (Figure 1). At the same time, existing problems are identified and possible future research directions are suggested.

Figure 1 The research ideas and methods of disassembled prescriptions study

Single herb or mineral study

A single herb or mineral study refers to investigations of the pharmacological action of every active ingredient in a prescription and the identification of the primary active ingredients. A single herb or mineral study can help to identify drugs that play a major role in a formula.

Xi‑Huang (XH) formula, a prescription that has been used since the 18th century to treat various diseases, is widely used for tumor therapy alone or in combination with chemotherapy and/or radiotherapy. XH formula is composed ofCalculus Bovis,Moschus,Olibanum, andMyrrha[7]. Our team used molecular biology and bioinformatics methods to explore the antiestrogenic activity of the XH formula [8]. We first used the crude extract of XH formula and single drug components to observe the effects on the viability of breast cancer cell lines. The results showed that the half‑maximal inhibitory concentrations ofCalculus BovisandMoschuswere higher than those ofCommiphora myrrhaandOlibanum. Therefore,MoschusandCalculus Bovismay not be responsible for the direct inhibitory effects of XH.Through a single herb study, we found thatCommiphora myrrhaandOlibanumwere the main anti‑breast cancer herbs in the XH formula.In addition, our study confirmed the anti‑estrogen receptor+ breast cancer effects of XH formula in vitro and in vivo,and the effects of XH formula were stronger compared to those of every herb that constituted XH individually.

Single herb or mineral studies help distinguish between primary and secondary herbs or minerals. However, this method ignores the interactions between the different herbs.

Sub-prescription study

Generally, the prescriptions of TCM are composed of more than two herbs or minerals.Therefore,a large formula may contain one or more sub‑prescriptions. When studying these large formulae,sub‑prescriptions are commonly separated into their individual groups for further study.

Erlong Zuoci pill(ELZCP), one of the most popular classical Chinese herbal prescriptions, is widely used clinically in patients with tinnitus and deafness of kidney deficiency syndrome [9]. ELZCP is composed of eight Chinese medicines: Rhizome ofAdhesive Rehmannia,Fructus Corni,Rhizoma Dioscoreae,Poria,Rhizoma Alismatis,Cortex Moutan,Radix Bupleuri, andMagnetitum[10]. All of these, excludingRadix BupleuriandMagnetitum, constitute a prescription called the Liuwei Dihuang pill [11]. Therefore, in a previous study, the prescriptions were divided into ELZCP, Liuwei Dihuang, Chaihu Cishi, and eight single herbal groups. The results showed that the protective effect of ELZCP on the cells against gentamicin damage was the best, and the Liuwei Dihuang group also showed a lesser protective effect.However, the Chaihu Cishi group did not protect cells against gentamicin damage, but strengthened the protective effect of Liuwei Dihuang. In Chinese medicine,MagnetitumandRadix Bupleurican be considered as“assistant herbs”.These results suggested the rationality of the compatibility of ELZCP and reflected the advantages of the compatibility of the composition of Chinese medicinal prescriptions.

The sub‑prescription method helps identify groups of herbs with effective actions and partly considers the interactions between different herbs. However, this method is inapplicable for small amounts of formula, as there are too few herbs for further grouping.

Grouping based on the property and flavor of Chinese medicines

The theory of Chinese herbal medicine properties, mainly property and flavor (four properties and five tastes), meridial distribution,ascending‑descending‑floating‑sinking, and toxicity, is an important part of TCM theory. Property and flavor theory is the core part of the Chinese herbal medicine property theory. There is an intrinsic relationship between the properties and flavors of herbs and their functions. In general, if several Chinese medicines have similar properties and flavors, they can treat similar diseases and syndromes.Therefore, these medicines can be grouped together for the study of disassembling prescriptions.

Liu et al. explored the effects ofPinelliadecoction, a prescription fromTreatise on Febrile Diseaseswritten by Zhang Zhongjing from the Han Dynasty (202 B.C.E.–220 C.E.), and its disassembled herb groups on the malignant transformation of bone marrow mesenchymal stem cells (BMSCs) in the gastric cancer microenvironment [12]. In this study, the authors divided subjects into six groups, including four groups treated with Chinese medicine recipes: (A) full recipe group:Banxia (Rhizoma Pinelliae), Huangqin (Radix Scutellariae), Ganjiang(Rhizoma Zingiberis), Renshen (Radix Ginseng), Zhigancao (Radix Glycyrrhizae Uralensis), Huanglian (Rhizoma Coptidis), and Dazao(Fructus Jujubae); (C) bitter drugs for purgation group: Huangqin(Radix Scutellariae) and Huanglian (Rhizoma Coptidis); (D)sweet‑nourishing group: Renshen (Radix Ginseng), Dazao (Fructus Jujubae), and Zhigancao (Radix Glycyrrhizae Uralensis). After co‑culture with gastric cancer BGC‑823 cells, BMSC proliferation and migration were inhibited in all four drug groups.The BMSC inhibitory effect in groups B and C was not significantly different from that in group A. Moreover, the inhibitory effect in group B was stronger than that in other treatment groups [12]. However, the authors did not further discuss why the disassembled herb groups had similar or even stronger effects when used as part of the full prescription on BMSCs after co‑culture with BGC‑823 cells.

To some extent, the properties and flavors of Chinese medicines reflect the commonality between different herbs, which is closely related to their chemical constituents. Thus, grouping based on the properties and flavors of Chinese medicine has a scientific basis.However, property and flavor are only two characteristics of a herb,and there is bias for grouping based on these factors alone.

Grouping according to the similar efficacy of Chinese medicines

There are 19 species of herbs, including diaphoretic herbs, antipyretic herbs, and purgatives, according to the function of Chinese medicines in Chinese materia medica. Grouping according to the similar efficacy of Chinese medicines refers to medicines being combined into a subgroup based on their similar contributions, which usually provide patients with synergistic benefits by acting on common biological targets or pathways.

Wang et al. used a liver injury model to explore the effects of the Bushen recipe (BS) and its disassembled prescriptions on the toll‑like receptor 3/9 signaling pathway [13]. The BS was divided into four subgroups. The first was the whole BS group, which was comprised ofHerba Epimedii,Cuscuta ChinensisLam,Cistanche DeserticolaMa,Fallopia Multiflora,Radix RehmanniaeRecen,Lycium Barbarum,Giant Knotweed Rhizome,Radix Scutellariae,Radix Sophorae Flavescentis, andPericarpium Citri Reticulatae Viride. The second group was the Bushen‑Yang group, which comprisedHerba Epimedii,Cuscuta ChinensisLam, andCistanche DeserticolaMa. The third group was the Bushen‑Yin group, which comprisedFallopia Multiflora, Radix RehmanniaeRecen, andLycium Barbarum. The fourth group was the Qinghua group, which comprised giantKnotweed Rhizomes,Radix Scutellariae, andRadix Sophorae Flavescentis. The results showed that the BS group was a more effective treatment than the other disassembled prescription groups, which provided evidence for the whole Bushen recipe use for the treatment of liver injury. The study also demonstrated the compatibility of Chinese prescriptions.

Shenghui soup is a TCM formula used clinically to treat forgetfulness. This formula can be divided into four subgroups: the enriching yin and nourishing the kidney group (Dihuang (Rehmannia glutinosa)and Shanzhuyu(Cornus officinalis));the nourishing the heart and tranquilizing the mind group (Dazao (Fructus Jujubae) and Cebai(Platycladus orientalis)); the tonifying Qi (In traditional Chinese medicine, it refers to the most fundamental and subtle substances that constitute the human body and maintain life activities. At the same time, it also has the meaning of physiological function. In terms of traditional Chinese medicine,Qi and different words are used together to express different meanings.) and strengthening the spleen group(Fuling (Poria cocos) and Renshen (Radix Ginseng)); and the eliminating phlegm and strengthening intelligence group (Yuanzhi(Polygala tenuifolia), Cangpu (Acorus calamus), and Baijiezi (Sinapis alba). In this formula, the different subgroups may have different effects. Neurite outgrowth and differentiation of PC12 cells were significantly inhibited by the enriching yin and nourishing kidney group, while the tonifying qi and strengthening spleen group and eliminating phlegm and strengthening intelligence group played opposite roles [14, 15].

The two studies mentioned above present representative formulae for dissection research grouped according to the efficacy of Chinese medicines. However, the efficacy of Chinese medicines lacks quantitative criteria and fails to achieve precise classification.

Active ingredients or effective parts study of a formula

Multiple components can act on multiple targets and exert synergistic therapeutic efficacies in some formulae [16, 17]. However, both the essential compounds and the precise mechanisms of action of most formulae remain to be addressed by molecular biology methods. As a result,some studies have used a representative component of one herb or mineral from one formula to explore the compatibility principles and precise action mechanisms of the entire formula.

A famous study conducted by Chen et al. used the effective constituents of realgar,Indigo naturalis, andSalvia miltiorrhizato confirm the anti‑promyelocytic leukemia effects of Realgar‑Indigo naturalis [18]. In the study, the main components of Realgar‑Indigo naturalis were realgar,Indigo naturalis, andSalvia miltiorrhiza, with tetraarsenic tetrasulfide (A), indirubin (I), and tanshinone IIA (T) as the major active ingredients. Finally, the study indicated that the whole prescription yielded synergy in the treatment of acute promyelocytic leukemia (APL) using a murine APL model in vivo and APL cell differentiation induction in vitro. The study also illuminated the “Jun‑Chen‑Zuo‑Shi” compatibility relationship, where A is the principal component of the formula and T and I serve as adjuvant ingredients.

Another study dissected the mechanisms of Danhong injection(DHI) in myocardial ischemia/reperfusion injury. DHI is a standardized water‑soluble complex containing extracts ofRadix Salvia miltiorrhizaandFlos Carthamus tinctoriusL. [19]. First, four ingredients were identified fromSalvia miltiorrhizaand one fromCarthamus tinctoriusfrom DHI using HPLC fingerprinting. Finally, the effects of hydroxysafor yellow A (A), salvianolic acid B (B), and danshensu (C) on myocardial ischemia/reperfusion injury in rats and cardiomyocytes were assessed. The results showed that yellow A exerted a more prominent anti‑inflammatory effect than salvianolic acid B and danshensu; salvianolic acid B had a more powerful antioxidative capacity than hydroxysafor yellow A and danshensu;and danshensu had stronger antiapoptotic ability than hydroxysafor yellow A and salvianolic acid B,which indicated that the DHI formula exerts its therapeutic polypharmacology through systematic drug combinations acting on multiple targets.

The study provided a relatively accurate method for clarifying the primary ingredients or parts of a formula. One the one hand,compared with the methods mentioned above, this method further explored the primary ingredients for one or more whole herbs.However, active ingredients or effective parts are partially representative, which may lead to biased conclusions.

A model to study large amounts of formulae

Clinically, certain formulae are composed of dozens of herbs or minerals used to treat difficult miscellaneous diseases. Compared to small formulae, the study of large formulae is more difficult.

Prof. Xiong‑Zhi Wu et al. used an ancient formula called Hua‑Zheng‑Hui‑Sheng‑Dan, which includes 35 Chinese medicines, as an experimental model and established a research method for large formulae. First, the authors conducted single‑herb screening using molecular biology approaches to determine their antitumor effects.They then used the median effect principle and orthogonal experimental design to further optimize and reduce the large formula into a small formula and, finally, obtained a small formula called samll Hua‑Zheng‑Hui‑Sheng‑Dan. More importantly, the optimized samll Hua‑Zheng‑Hui‑Sheng‑Dan exhibited superior antitumor activity compared with the original formula both in vitro and in vivo[20].

The study of large formula dissections usually requires multiple approaches simultaneously, even mathematical methods, to explore compatibility theory. Another aim for large‑formula dissection is to downsize and develop a new formula based on the guarantee of effectivity.

Systems pharmacology study

In addition to classical research methods for disassembled prescription studies, the emergence and development of network pharmacology has made TCM formula studies more systematized and integrated. An increasing number of studies have used network pharmacology and celled systems pharmacology to dissect the relationship between TCM formulae and diseases [21–24].

In 2007, British scientist Hopkins proposed a new discipline concept, network pharmacology, which combines pharmacology with network analysis. The concept is based on multidisciplinary theories,such as systems biology and multidirectional pharmacology [25].Network pharmacology, also known as systems pharmacology,provides a new method to uncover the complex action mechanisms of TCM in complicated diseases by integrating systems biology and pharmacology, including pharmacokinetic evaluation, target prediction, and network/pathway analysis [26, 27]. Therefore, the network pharmacology concept naturally links back to TCM formulae,which consist of combinations of herbs where multiple active phytochemical components may theoretically target multiple targets/pathways [28]. Network pharmacology combined with experimental methodologies could further verify the predicted targets and active ingredients of the TCM formulae (Figure 2). Therefore,network pharmacology is a good tool for dissecting TCM formulae and modernizing TCM. Indeed, the annual volume of TCM network pharmacology publications has shown an increasing trend [29]. This strategy of dissecting the complexity of clinically effective formulae at the chemical, molecular, cellular, and organism levels may be an effective way to explore the value of TCM [30]. More importantly,based on this, network relationships among active components,targets, and diseases have been built to uncover the pharmacological actions of TCM formulae. In other words, the results of network pharmacology fully demonstrate not a single agent or a single target,but the holistic therapeutic philosophy of TCM formula.

Figure 2 The model of systems pharmacology study. TCM,traditional Chinese medicine; ADME, absorption, distribution,metabolism, excretion; GO/KEGG, Gene Ontology/Kyoto Encyclopedia of Genes and Genomes.

Several problems that must be addressed

TCM formulae are complex herbal mixtures that target multiple etiological factors simultaneously,which makes them difficult to study thoroughly. As mentioned above, TCM practitioners, pharmacy specialists, and bioinformatics experts have made great efforts to explore the mechanisms of action of TCM formulae. However, several problems must be addressed. First, new compositions may possibly come into being, and the content of active ingredients may change during the decoction process of compound prescription. Thus, the newly produced compositions and content changes should be considered in disassembled prescription studies to fully explore the underlying mechanisms of TCM formulae. Unfortunately, the identification of new produced compositions and content changes is difficult. Second, in addition to the Chinese formula decoction, there are many other dosage forms, such as powder, pill, and mastic. After using different processing methods, some herbs or miners may produce new components with different effects. Therefore,disassembled prescription studies should consider processing factors.Third, the pharmacokinetics of oral TCM formulae are quite complicated and still unclear. Fourth, mineral Chinese medicines are usually difficult to dissolve in water, thus, such medicines are commonly excluded from experiments.

Conclusion and perspective

To modernize and generalize TCM formulas for treating diseases,there is an urgent need to specify the precise action mechanisms of TCM formulae. Disassembled prescription studies using modern molecular biology approaches provide a reasonable research strategy to explore the compatibility theory and scientific connotation of TCM formulae.In this review, several main study methods for formula dissection were summarized and it was found that several methods are usually used together in one study. However, there are some problems that must be resolved urgently to improve the research quality and fully identify the underlying mechanisms of TCM formulae.